Matt:LabNotes/2014-9-8: Difference between revisions
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>Mzcai (Created page with "==in situ Padlock Probe Capture Temp: 45C vs 60C== ===Procedure=== *PGP1F Samples with 1st Rolonies prepared by Hosuk **Fixed 9/6/14 and 1st Rolonies on 9/8/14 *Add 100ul Pa...") |
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*Incubate at 37C for 30min | *Incubate at 37C for 30min | ||
*Incubate at '''45C or 60C''' for | *Incubate at '''45C or 60C''' for 26hrs | ||
*Wash with 1X PBS twice | |||
*Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (pre-heated to 75C) and incubate at 45C for 30min | |||
*Wash with 2X SSC twice | |||
*Add 200ul RCA Mix and incubate at 30C for 16hrs | |||
**H2O : 172uL | |||
**10x Buffer : 20uL | |||
**dNTP : 2uL | |||
**a-dUTP : 4uL | |||
**Phi29 (100 U/µl) : 2uL | |||
*Wash with 1X PBS once | |||
*Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr | |||
*Wash with 1X PBS twice | |||
*Add 200ul 1M Tris pH 8.0 at RT for 30min | |||
*Wash with 1X PBS twice and store in PBS | |||
===Imaging=== | |||
Dish #1 (45C) | |||
20X Objective | |||
Laser 4% | |||
Gain 680 | |||
Offset -1.05% | |||
Pinhole 1.00 | |||
Zoom 1.00 | |||
4096x4096 | |||
600Hz | |||
Pos1: (67.8,41.12) z: 1552.95-1572.66 | |||
Pos2: (67.31,42.03)z: 1559.43-1578.20 | |||
Pos3: (65.91,40.54)z: 1565.21-1585.86 | |||
Dish #2 (60C) | |||
Pos1: (71.5,40.12) z: 1361.76-1380.53 | |||
Pos2: (71.02,45.03) z: 1313.81-1334.46 | |||
===Result=== | |||
*Using PISA to count features | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''45C''' | |||
| align="center" style="background:#f0f0f0;"|'''45C''' | |||
| align="center" style="background:#f0f0f0;"|'''60C''' | |||
| align="center" style="background:#f0f0f0;"|'''60C''' | |||
|- | |||
| ||1st Rolonies||COL1A1 Rolonies||1st Rolonies||COL1A1 Rolonies | |||
|- | |||
| Position 1||3,268||1,887 (58%)||1,751||1,973 (113%) | |||
|- | |||
| Position 2||4,341||2,259 (52%)||2,342||1,820 (78%) | |||
|- | |||
| Position 3||3,891||1,609 (41%)|||| | |||
|} | |||
====Example images==== | |||
[[File:20140911_45C_Pos2_1stRolonies.PNG]] [[File:20140911_45C_Pos2_COL1A1Rolonies.PNG]] | |||
===Conclusion=== | |||
*60C may be more effective for Ampligase step but hard to compare because there were a different number of starting primary rolonies | |||
**Since 60C is definitely not worse we should continue with 60C padlock probe capture until a problem occurs | |||
*ppCOL1A1 is highly localized to nucleus, even though primary rolonies are evenly distributed in cell |
Latest revision as of 02:58, 28 September 2014
in situ Padlock Probe Capture Temp: 45C vs 60C[edit]
Procedure[edit]
- PGP1F Samples with 1st Rolonies prepared by Hosuk
- Fixed 9/6/14 and 1st Rolonies on 9/8/14
- Add 100ul Padlock Probe Mix
Components | Volume |
5 U/ul Ampligase | 10 |
10X Ampligase Buffer | 10 |
100uM ppCOL1A1 | 2 |
H2O | 78 |
Total | 100 |
- Incubate at 37C for 30min
- Incubate at 45C or 60C for 26hrs
- Wash with 1X PBS twice
- Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (pre-heated to 75C) and incubate at 45C for 30min
- Wash with 2X SSC twice
- Add 200ul RCA Mix and incubate at 30C for 16hrs
- H2O : 172uL
- 10x Buffer : 20uL
- dNTP : 2uL
- a-dUTP : 4uL
- Phi29 (100 U/µl) : 2uL
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr
- Wash with 1X PBS twice
- Add 200ul 1M Tris pH 8.0 at RT for 30min
- Wash with 1X PBS twice and store in PBS
Imaging[edit]
Dish #1 (45C)
20X Objective Laser 4% Gain 680 Offset -1.05% Pinhole 1.00 Zoom 1.00 4096x4096 600Hz
Pos1: (67.8,41.12) z: 1552.95-1572.66 Pos2: (67.31,42.03)z: 1559.43-1578.20 Pos3: (65.91,40.54)z: 1565.21-1585.86
Dish #2 (60C)
Pos1: (71.5,40.12) z: 1361.76-1380.53 Pos2: (71.02,45.03) z: 1313.81-1334.46
Result[edit]
- Using PISA to count features
' | 45C | 45C | 60C | 60C |
1st Rolonies | COL1A1 Rolonies | 1st Rolonies | COL1A1 Rolonies | |
Position 1 | 3,268 | 1,887 (58%) | 1,751 | 1,973 (113%) |
Position 2 | 4,341 | 2,259 (52%) | 2,342 | 1,820 (78%) |
Position 3 | 3,891 | 1,609 (41%) |
Example images[edit]
File:20140911 45C Pos2 1stRolonies.PNG File:20140911 45C Pos2 COL1A1Rolonies.PNG
Conclusion[edit]
- 60C may be more effective for Ampligase step but hard to compare because there were a different number of starting primary rolonies
- Since 60C is definitely not worse we should continue with 60C padlock probe capture until a problem occurs
- ppCOL1A1 is highly localized to nucleus, even though primary rolonies are evenly distributed in cell